This proposal focuses on the structural and functional characterization of the hepatocyte nuclear factor 4 alpha (HNF4a), a master transcriptional regulator of metabolic gene programs. HNF4a does not respond to ligand binding, but instead appears to rely on methylation and phosphorylation, and on the induction of coactivators for regulating its transcriptional activity. Our recent structural characterization of HNF4a reveals multiple domain-domain interactions that were unanticipated. A highly interconnected domain arrangement links subunit dimerization to both DNA and coactivator binding. We wish to validate this understanding of the receptor's allosteric arrangement using a series of biochemical and cell-based studies that probe these inter-domain connections. We also found several posttranslational modifications that map to key domain-domain junctions. Therefore, a second goal is to understand the allosteric mechanism by which covalent modifications are transmitted to the DNA binding domain of the receptor. The third goal is to expand our structural and mechanistic understanding of how coactivators interact with HNF4a. Here we will test a novel hypothesis that some coactivators, including PGC-1a, can physically drive and stabilize the productive complex of HNF4a on DNA. PUBLIC HEALTH RELEVANCE: The studies we propose are focused on HNF4a, a master regulator of genes in the liver and in pancreatic islet cells. HNF4a regulates the production of glucose and insulin, and has been linked to a form of diabetes. To understand the way in which HNF4a functions, we will apply techniques that allow us visualize its protein architecture, and examine its interactions with other proteins.